Attempts to repair the mammalian brain or replace CNS functions resulting from defects or following disease or damage to the CNS are hampered by an incomplete understanding of the complex structure-function relationships in the CNS. Although knowledge of some basic principles of cell function in the brain has advanced greatly in recent years, understanding of interactions between clusters of cells or systems and cell circuits in different regions of the brain and their relationship to the outward manifestations of behavior and neurological function lags far behind. Difficulties in approaching these problems have been caused, in part, by the large number of different cell types in the mammalian CNS and the number and complexity of their connections. In addition, the blood-brain barrier makes access to the brain for diagnosis, treatment and the design of new therapies more difficult.
In spite of the absence of sophisticated knowledge of pathophysiology of most normal or abnormal brain functions, some attempts at pharmacological therapy for CNS dysfunction have already become useful and effective. These include the use of psycho-active drugs for psychiatric disorders such as schizophrenia, and specific replacement therapy in the rare cases in which the biochemical and cellular bases of the CNS disorder are relatively better understood, as in Parkinson's disease. At the core of most therapeutic approaches is the objective of replacing or reactivating a specific chemical function in the brain that has been lost as a consequence of disease or damage.
Intracerebral neural grafting has emerged recently as an additional potential approach to CNS therapy. The replacement or addition of cells to the CNS which are able to produce and secrete therapeutically useful metabolites may offer the advantage of averting repeated drug administration while also avoiding the drug delivery complications posed by the blood-brain barrier. (Rosenstein, Science 235:772-774 (1987)). While the concepts and basic procedures of intracerebral grafting have been known for decades, most of the factors that optimize the survival of grafted cells have only recently come to be investigated and partially understood. (Bjorklund et al., in Neural Graftinc in the Mammalian CNS, p. 709, Elsevier, Amsterdam (1985); Sladek et al., in Neural Transplants: Development and Function, Plenum Press, N.Y. (1984)). Several factors critical for reliable and effective graft survival have been identified, including the following:
(1) Age of the donor: efficiency of grafting is reduced with increasing age of donor cells.
(2) Age of the host: young recipients accept grafts more readily than older ones.
(3) Availability of neuronotrophic factors in the host and donor tissue: wound-induced neurotrophic factors enhance graft survival.
(4) Immunological response: the brain is not totally an immunologically privileged site.
(5) The importance of target-donor matching: neurons survive better when they are grafted to a site which they would normally innervate.
(6) Vascularization: it is critical that the grafts be vascularized rapidly or otherwise sufficiently well nourished from the environment.
As these critical factors have become recognized and optimized, intracerebral grafting has become a valid and reliable tool for neurobiologists in the study of CNS function and potentially for clinicians for the design of therapies of CNS disease. This approach has reached a level of experimental clinical application in Parkinson's disease.
Parkinson's disease is an age-related disorder characterized by a loss of dopamine neurons in the substantia-nigra of the midbrain, which have the basal ganglia as their major target organ. The symptoms include tremor, rigidity and ataxia. The disease is progressive but can be treated by replacement of dopamine through the administration of pharmacological doses of the precursor for dopamine, L-DOPA, (Marsden, Trends Neurosci. 9:512 (1986); Vinken et al., in Handbook of Clinical Neurology p. 185, Elsevier, Amsterdam (1986)), although with chronic use of pharmacotherapy the patients often become refractory to the continued effect of L-DOPA. There are many suggested mechanisms for the development of the refractory state, but the simplest is that the patients reach a threshold of cell loss, wherein the remaining cells cannot synthesize sufficient dopamine from the precursor.
Parkinson's disease is the first disease of the brain for which therapeutic intracerebral grafting has been used in humans. Several attempts have been made to provide the neurotransmitter dopamine to cells of the diseased basal ganglia of Parkinson's patients by homografting adrenal medullary cells to the brain of affected patients. (Backlund et al., J. Neurosurc. 62:169-173 (1985); Madrazo et al., New Eng. J. Med. 316:831-836 (1987)). The transplantation of other donor cells such as fetal brain cells from the substantianigra, an area of the brain rich in dopamine-containing cell bodies and also the area of the brain most affected in Parkinson's disease, has been shown to be effective in reversing the behavioral deficits induced by selective dopaminergic neurotoxins. (Bjorklund et al., Ann. N.Y. Acad. Sci. 457:53-81 (1986); Dunnett et al., Trends Neurosci. 6:266-270 (1983)). These experiments suggest that synaptic connectivity may not be a requisite for a functional graft and that it may be sufficient to have cells constitutively producing and secreting dopamine in the vicinity of the defective cells.
With this background, it seems likely that Parkinson's disease is a candidate disease for the transplantation of genetically engineered cells, because (1) the chemical deficit is well known (dopamine), (2) the human and rat genes for the rate-limiting enzyme in the production of dopamine have been cloned (tyrosine hydroxylase), (3) the anatomical localization of the affected region has been identified (basal ganglia), and (4) synaptic connectivity does not appear to be required for complete functional restoration.
The recent demonstration of genetic components in a rapidly growing list of other CNS diseases, including Huntington's disease, (Gusella et al., Nature 306:234-238 (1983)) Alzheimer's disease, (Delabar et al., Science, N.Y. 235;1390-1392 (1987); Goldgaber et al., Science, N.Y. 235:877-880 (1987); St. George-Hyslop et al., Science, N.Y. 235:885-890 (1987); Tanzi et al., Science, N.Y. 235:880-884 (1987); bipolar disease (Baron et al., Nature 326:289-292 (1987)); schizophrenia (Sherrington et al., Nature 336:164-167 (1988) and many other major human diseases, suggests that these other CNS diseases will eventually become accessible to gene therapy approaches.
In parallel to the progress in neurobiology during the past several decades, advances in an understanding of molecular biology and the development of sophisticated molecular genetic tools have provided new insights into human disease in general. As a result, medical scientists and geneticists have developed a profound understanding of many human diseases at the biochemical and genetic levels. The normal and abnormal biochemical features of many human genetic diseases have become understood, the relevant genes have been isolated and characterized, and early model systems have been developed for the introduction of functional wild-type genes into mutant cells to correct a disease phenotype. (Anderson, Science 226:401-409 (1984)). The extension of this approach to whole animals, that is, the correction of a disease phenotype in vivo through the use of the functional gene as a pharmacologic agent, has come to be called "gene therapy". (Friedmann et al., Science 175:949-955 (1972); Friedmann, Gene Therapy Fact and Fiction, Cold Spring Harbor Laboratory, N.Y. (1983)). Gene therapy is based on the assumption that the correction of a disease phenotype can be accomplished either by modification of the expression of a resident mutant gene or the introduction of new genetic information into defective or damaged cells or organs in vivo.
At present, techniques for the ideal versions of gene therapy, that is through site-specific gene sequence correction or replacement in vitro, are just beginning to be conceived but are not yet well developed. Therefore, most present models of gene therapy are actually genetic augmentation rather than replacement models and rely on the development of efficient gene-transfer systems to introduce functional, wild-type genetic information into genetically defective cells in vitro and in vivo. To be clinically useful, the availability of efficient delivery vectors for foreign DNA sequences (transgenes) must be combined with easy accessibility of suitable disease-related target cells or organs and with the development of techniques to introduce the vector stably and safely into those target cells.
Model systems for the genetic and phenotypic correction of simple enzymatic deficits are now being developed and studied, as is the identification of the appropriate potential recipient cells and organs associated with specific metabolic and genetic diseases. Evidence has recently been obtained to show that foreign genes introduced into fertilized mouse eggs can correct disease phenotype. (Constantini et al., Science 233:1192-1194 (1986); Mason et al., Science 234:1372-1378 (1986); and Readhead et al., Cell 48:703-712 (1987)).
A great deal of attention has recently been paid to the use of gene delivery vectors derived from murine retroviruses (Anderson, Science 226:401-409 (1984); Gilboa et al, Biotechniques 4:504-512 (1986)) for gene transfer at later stages of development. Gene transfer in vitro using such retroviral vectors is extremely efficient for a broad range of recipient cells, the vectors have a suitably large capacity for added genes, and infection with them does little metabolic or genetic damage to recipient cells. (Shimotohno et al., Cell 26:67-77 (1981); Wei et al., J. Virol. 39:935-944 (1981); Tabin et al., Molec. Cell. Biol. 2:426-436 (1982)). Several useful systems have demonstrated that the expression of genes introduced into cells by means of retroviral vectors can correct metabolic aberrations in vitro in several human genetic diseases associated with single-gene enzyme deficiencies. (Kantoff et al., Proc. Nat'l. Acad. Sci. USA 83:6563-6567 (1986); Willis et al., J. Biol. Chem. 259:7842-7849 (1984)). There has been particular interest in bone marrow as a potential target organ for this approach to gene therapy because of the prevalence and importance of disorders of bone marrow-derived cell lineages in a variety of major human diseases, including the thalassemias and sickle-cell anemia, Gaucher's disease, chronic granulomatous disease (CGD) and immunodeficiency disease resulting from deficiencies of the purine pathway enzymes, adenosine deaminase (ADA) and purine nucleoside phosphorylase (PNP) (Kantoff, supra; McIvor et al., Molec. Cell. Biol. 7:838-846 (1987); Soriano et al., Science 234:1409-1413 (1986); Willis et al., supra)). Other metabolically important target organs, such as the liver, have also recently become theoretically susceptible for genetic manipulation through the demonstration of infection of cells from such organs with viral vectors (Wolff et al., Proc. Nat'l Acad. Sci. USA 84:3344-3348 (1987)). Furthermore, the discovery of numerous cell-specific regulatory signals such as cis-acting enhancers, tissue-specific promoters and other sequences may provide tissue specific gene expression in many other organs even after general, non-specific infections and gene transfer in vivo (Khoury et al., Cell 33:313-314 (1983); Serflin et al., Trends Genet. 1:224-230 (1985)).
A recently developed model of gene therapy uses target cells removed from a subject, placed in culture, genetically modified in vitro, and then re-implanted into the subject (Wolff et al., Rheumatic Dis. Clin. N. Amer. 14(2):459-477 (1988); Eglitis et al. Biotechnicues 6:608-614 (1988); Ledley, J. Pediatrics 110:1-8 (1987)). Target cells have included bone marrow stem cells (Joyner et al., Nature 305:556-558 (1983); Miller et al., Science 225:630-632 (1984); Williams et al., Nature 310:476-480 (1984)); fibroblasts (Selden et al., Science 236:714-718 (1982); Garver et al., Proc. Nat'l. Acad. Sci. USA 84:1050-1054 (1987) and St. Louis et al., Proc. Nat'l. Acad. Sci. USA 85:3150-3154 (1988)), keratinocytes (Morgan et al., Science 237:1476-1479 (1987)) and hepatocytes (Wolff et al., Proc. Nat'l. Acad. Sci. USA 84:3344-3348 (1987)). This indirect approach of in vivo gene transfer is necessitated by the inability to transfer genes efficiently directly into cells in vivo. Although there has been some recent progress towards genetically modifying neurons in culture (Geller et al., Science 241:1667-1669 (1988)), this indirect approach of in vivo gene transfer has not yet been applied to the CNS.
There are several ways to introduce a new function into target cells in the CNS in a phenotypically useful way i.e. to treat defects, disease or dysfunction (FIG. 1). The most direct approach, which bypasses the need for cellular grafting entirely, is the introduction of a transgene directly into the cells in which that function is aberrant as a consequence of a developmental or genetic defect, i.e. neuronal cells in the case of Tay-Sachs disease, possibly Lesch-Nyhan disease, and Parkinson's disease (1, in FIG. 1). Alternatively, a new function is expressed in defective target cells by introducing a genetically modified donor cell that could establish tight junction or other contacts with the target cell (2, in FIG. 1). Some such contacts are known to small molecules from one cell to another, leading to phenotypic changes in the recipient cell (Lowenstein, Biochim. Biochys. Actg. 560:1-66 (1979)). This process has been called "metabolic co-operation" and is known to occur between fibroblasts and glial cells (Gruber et al., Proc. Nat'l. Acad. Sci. USA 82:6662-6666 (1985)), although it has not yet been demonstrated conclusively in neurons. Still other donor cells could express and secrete a diffusible gene product that can be taken up and used by nearby defective target cells (3, in FIG. 1). The donor cells may be genetically modified in vitro or alternatively they may be directly infected in vivo (4, in FIG. 1). This type of "co-operativity" has been demonstrated with CNS cells, as in the case of NGF-mediated protection of cholinergic neuronal death following CNS damage (Hefti, J. Neurosci. 6:2155 (1986); Williams et al., Proc. Nat'l. Acad. Sci. USA 83:9231-9235 (1986)). Finally, an introduced donor cell infected with not only replication-defective vector but also replication-competent helper virus, could produce locally high liters of progeny virus that might in turn infect nearby target cells to provide a functional new transgene (5, in FIG. 1).
There are several types of neurons in the mammalian brain. Cholinergic neurons are found within the mammalian brain and project from the medial septum and vertical limb of the diagonal band of Broca to the hippocampal formation in the basal forebrain. The short, nerve-like portion of the brain connecting the medial septum and vertical limb of the diagonal band with the hippocampal formation is termed the "fimbria fornix". The fimbria fornix contains the axons of the neurons located in the medial septum and diagonal band. An accepted model of neuron survival in vivo is the survival of septal cholinergic neurons after fimbria fornix transection or lesion (also termed "axotomy"). Axotomy severs the cholinergic neurons in the septum and diagonal band and results in the death of up to one-half of the cholinergic neurons (Gage et al., Neuroscience 19:241-256 (1986)). This degenerative response is attributed to the loss of trophic support from nerve growth factor (NGF), which is normally transported retrogradely in the intact brain from the hippocampus to the septal cholinergic cell bodies (Korsching et al., Proc. Nat'l. Acad. Sci. USA 80:3513 (1983); Whittemore et al., Proc. Nat'l. Acad. Sci. USA 83:817 (1986); Shelton et al., Proc. Nat'l Acad. Sci USA 83:2714 (1986); Larkfors et al., J. Neurosci. Res. 18:525 (1987); and Seilor et al., Brain Res. 300:33 (1984)).
Studies have shown that chronic intra-ventricular administration of NGF before axotomy will prevent cholinergic neuron death in the septum (Hefti, J. Neurosci. 8:2155-2162 (1986); Williams et al., Proc. Nat'l Acad. Sci. USA 83:9231 (1986); Kromer, Science L 235 214 (1987); Gage et al., J. Comp. Neurol. 269:147 (1988)). Fimbria fornix transection thus provides an in vivo model for determining at various points in time the ability of various therapies to prevent retrograde neuronal death.
It would be advantageous to develop procedures for gene transfer via efficient vectors followed by intracerebral grafting of the genetically modified cells in vivo to treat disorders of the CNS.